Natural immunostimulant compositions, methods for obtaining the same and pharmaceutical formulations thereof

ABSTRACT

A natural immunostimulant composition for the treatment of immunodeficiency, the composition comprising extract of plant  Symplocos racemosa  and/or plant  Prosopis glandulosa  and a pharmaceutically acceptable carrier is disclosed. Also disclosed are methods for obtaining the plant extract, methods for preparing the composition and methods of treating diseases related to immunodeficiency.

FIELD OF THE INVENTION

This invention relates to a natural immunostimulant composition, moreparticularly, a natural immunostimulant composition comprising extractof plant Symplocos racemosa and/or plant Prosopis glandulosa and apharmaceutically acceptable carrier, methods of obtaining the same,pharmaceutical formulations thereof and methods of treatingimmunodeficiencies in animals including humans using said naturalimmunostimulant composition.

BACKGROUND OF THE INVENTION

Immunomodulation is a process that alters the immune system of anorganism by interfering with its functions. This process results inimmunostimulation, an enhancement of immune reaction orimmunosuppression that results in the reduction of resistance againstinfections and stress, which could be caused due to environmental orchemotherapeutic factors.

As a therapeutic concept, immunostimulation has long been known inmedicine. It is defined as the injection of substances which themselveshave only weak, if any, antigenic effect, but are nevertheless able toinduce the body's own defense mechanisms in a specific or even nonspecific manner.

In the recent years, the research on immunomodulators has considerablyadvanced to form into a new field knowledge known as immunopharmacology.Immunomodulation is a strategy for overcoming incurable autoimmunediseases such as cancer, arthritis, allergies and AIDS. The immunesystem is supposed to provide both the theoretical and therapeuticdisorders of many chronic disorders. In today's world, extensiveexposure to pollutants and xenobiotics has resulted in the emergence ofatypical immune deficiencies that has changed the strategy for treatingpatients. Consequently, immunology has come to play a greater role.

To overcome the challenges posed by immunological deficiencies, majorresearch activities have been directed at developing new pharmaceuticalformulations, which are in turn aimed at modulating immune responses aswell as stimulating immune defense mechanisms. The recent research isalso aimed at safe and effective treatments for immunologicaldeficiencies.

RELATED ART

U.S. Pat. No. 6,548,086 to Maurya, et al., discloses a pharmaceuticalcomposition comprising Cryptolepis buchanani extract for treatingimmunodeficiencies in animals or human beings.

U.S. Pat. No. 6,413,553 to Bandyopadhyay, et al., discloses thepreparation of a pharmaceutical composition comprising an effectiveamount of lyophilised extracts of Piper betel and Murrya koeniggi asimmunomodulator.

U.S. Pat. No. 6,133,440 to Qiu, et al., discloses the preparation andisolation of biologically active polysaccharides from Aloe asimmunostimulating, immunomodulating and wound healing activities.

U.S. Pat. No. 6,030,622 to Shehadeh, et al., teaches the preparation ofa herbal extract composition comprising Arum, Pomegranate, Tea andHibiscus for the treatment of immune disorders and HIV infection.

U.S. Pat. No. 5,529,778 to Rohatgi, et al., discloses an ayurvedicformulation comprising Phyllanthus niruri, Tinospora cordifolia,Phyllanthus emblica, Terminalia belerica and Terminalia chebula etc, forprophylaxis and treatment of AIDS, flu, TB and other immuno-deficiencyconditions.

U.S. Pat. No. 6,444,210 to Kournikakis, et al., discloses the effect ofbacterial polysaccharides on cell-mediated immunity in animals. Thispatent further discloses the use of such polysaccharides in preventingand treating various infections as well as in treating carcinomas.

SUMMARY OF THE INVENTION

It is the principal aspect of the present invention to disclose theimmunopharmacological effects of the extracts of plant Symplocosracemosa and plant Prosopis glandulosa.

In another aspect, the present invention discloses the efficacy of theextracts of plants Symplocos racemosa and Prosopis glandulosa asimmunostimulator.

In still another aspect, the present invention provides for apharmaceutical composition containing a therapeutically effective amountof extracts of plants Symplocos racemosa and Prosopis glandulosa.

In yet another aspect, the present invention provides for apharmaceutical composition containing a therapeutically effective amountof extracts of plants Symplocos racemosa and Prosopis glandulosa or apharmaceutical composition comprising said extract of said plants, in apharmaceutically acceptable carrier or otherwise.

In one another aspect, the present invention provides for determiningthe role of a therapeutically effective amount of extracts of plantsSymplocos racemosa and Prosopis glandulosa in cell mediated immuneresponse.

In still another aspect, the present invention provides for determiningthe role of a therapeutically effective amount of extracts of plantsSymplocos racemosa and Prosopis glandulosa in humoral antibody response.

In yet another aspect, the present invention discloses methods oftreating immunodeficient patients.

It is also an aspect of the present invention to develop a method oftreating immunological disorders including AIDS, Hepatitis and Cancer.

In one another aspect, the present invention discloses methods ofproducing extracts from plant Symplocos racemosa and plant Prosopisglandulosa.

In one preferred embodiment, there is provided a natural immunostimulantcomposition comprising a therapeutically effective amount of the extractof plant Symplocos racemosa and plant Prosopis glandulosa, wherein theextract is prepared by all parts of said herb Prosopis glandulosa andpreferably its leaves.

In another preferred embodiment, there is provided a naturalimmunostimulant composition comprising a therapeutically effectiveamount of the extract of plants Symplocos racemosa and Prosopisglandulosa, wherein the extract is prepared by all parts of said herbSymplocos racemosa and preferably its bark.

In one another preferred embodiment, there is provided a naturalimmunostimulant composition comprising an aqueous extract of theequimolar mixture of coarse powders of leaves of plant Prosopisglandulosa and bark of plant Symplocos racemosa.

In yet another preferred embodiment, there is provided a naturalimmunostimulant composition comprising a therapeutically effectiveamount of extracts of plants Symplocos racemosa and Prosopis glandulosacomprising Alkaloids, Bitters, Glycosidic compounds, Tannins, Lipids,Polysaccharides, Flavonoids and Terpenoid glycosides as activeconstituents.

In one another preferred embodiment, there is provided a method ofobtaining the active fraction of extracts of plants Symplocos racemosaand Prosopis glandulosa by subjecting the extract to bioassay-guidedfractionation employing methanol soluble fraction, methanol and water(90:10) soluble fraction, methanol and water (75:25) soluble fraction,methanol and water (50:50) soluble fraction, methanol and water (25:75)soluble fraction and water-soluble fraction.

In still another preferred embodiment, there is provided a method ofobtaining the active fraction of extracts of plants Symplocos racemosaand Prosopis glandulosa by subjecting the extract to bioassay-guidedfractionation employing methanol soluble fraction and methanol:water(90:10) fraction.

In yet another preferred embodiment, there is provided a naturalimmunostimulant composition containing a therapeutically effectiveamount of extracts of plants Symplocos racemosa and Prosopis glandulosain a pharmaceutically acceptable carrier wherein the composition is inan oral dosage form.

In another preferred embodiment, there is provided a naturalimmunostimulant composition containing a therapeutically effectiveamount of extracts of plants Symplocos racemosa and Prosopis glandulosain an amount of 50 mg to 500 mg and pharmaceutically acceptable carrierscomprising sucrose (3.4 to 3.75 gm), Citric acid (0.01 to 0.02 mg),Methyl paraben sodium (0.01 mg), Propyl paraben sodium (0.0025 mg),Strawberry flavor (0.005 mg) and demineralised Water in a quantitysufficient to make up the formulation (Qs) to 5 ml of dosage form.

In yet another preferred embodiment, there is provided a naturalimmunostimulant composition comprising making granules containing atherapeutically effective amount of extracts of plants Symplocosracemosa and Prosopis glandulosa in an amount of 50 to 500 mg andpharmaceutically acceptable carriers comprising Microcrystallinecellulose (100 to 450 mg), pregelatinised Starch (about 50 mg), Lactose(50 to 300 mg), Dibasic calcium phosphate (50 to 250 mg), demineralisedWater in a quantity sufficient to make up the formulation (Qs) to 300 to900 mg of dosage form.

In another preferred embodiment, there is provided a naturalpharmaceutical composition comprising granules (500 to 900 mg) as perparagraph

and pharmaceutically acceptable excipients comprising Sodium starchglycolate (about 30 mg), Calcium carbonate (about 14 mg), colloidalsilicon dioxide (about 3 mg) and Magnesium stearate (about 3 mg) forfurther compression to obtain tablets.

In another preferred embodiment, there is provided a naturalpharmaceutical composition comprising granules (300 to 500 mg) as perparagraph and pharmaceutically acceptable excipients comprisingcolloidal silicon dioxide (about 2 mg) and Magnesium stearate (about 3mg) for further filling in capsules.

In one another preferred embodiment, there is provided a delivery systemcontaining natural immunostimulant composition wherein the deliverysystem comprises tablets, capsules, pills, granules and syrups, powders,concentrates, dry syrups etc.

In yet another preferred embodiment, there is provided a naturalimmunostimulant composition comprising a potency equivalent of theextract ranging from about 5 mg to about 500 mg.

In a still preferred embodiment, there is provided a method of treatingimmunodeficiencies by administering to a patient a naturalimmunostimulant composition comprising a therapeutically effectiveamount of extracts of plants Symplocos racemosa and Prosopis glandulosain a pharmaceutically acceptable carrier or otherwise.

In still another preferred embodiment, there is provided a naturalimmunostimulant composition, wherein the composition is used forstimulating both specific and non-specific immune reaction, enhancingresponsiveness of macrophages and subsets of T-lymphocytes andB-lymphocytes involved in antibody synthesis, stimulating humoralresponse, enhancing 4 fold (1:256) increases in the primary antibodytitre, increasing by 60% in phagocytosis, stimulating proinflammatorycytokines IL-6 at the rate of 1.4 times and TNF-α at the rate of 1.6times in mouse macrophage and fibroblast cell lines, suppressinginhibitory cytokines IL-10 in mice, treatment of AIDS patients,treatment of all types of cancers, treatment of Hepatitis and relapseconditions.

In still another preferred embodiment, there is provided a process forobtaining a natural immunostimulant composition, the process comprisingextracting Prosopis glandulosa leaves by percolation, filtering theplant extract, concentrating the plant extract to dryness on rotatoryevaporator or on steam bath at optimum temperature and producing aherbal composition comprising the said dry extract and pharmaceuticallyacceptable carrier.

In still another preferred embodiment of the present invention, there isprovided a process for preparation of a novel herbal composition. Themethod comprising, extracting plant extract from Prosopis glandulosa byhot soxhalation, filtering the plant extract, concentrating the plantextract to dryness on rotatory evaporator or on steam bath at optimumtemperature and producing a herbal composition employing the said dryextract and pharmaceutically acceptable carrier.

In still another preferred embodiment of the present invention, there isprovided a process for preparation of a novel herbal composition. Themethod comprising extracting plant extract from Symplocos racemosa bypercolation, filtering the plant extract, concentrating the plantextract to dryness on rotatory evaporator or on steam bath at optimumtemperature and producing a herbal composition employing the said dryextract and pharmaceutically acceptable carrier.

In still another preferred embodiment of the present invention, there isprovided a process for preparation of a novel herbal composition. Themethod comprising extracting plant extract from Symplocos racemosa byhot soxhalation, filtering the plant extract, concentrating the plantextract to dryness on rotatory evaporator or on steam bath at optimumtemperature and producing a herbal composition employing the said dryextract and pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects of the present invention together with additionalfeatures contributing thereto and advantages accruing therefrom will beapparent from the description of preferred embodiments of the presentinvention which are shown in the accompanying drawing figures.

FIG. 1 is a bar graph representation of the immunostimulant property ofIMS-6.

FIG. 2 is a bar graph representation of the effect of IMS-6 on NOproduction in RAW-264.7 Cells.

FIG. 3 is a bar graph representation of the effect of IMS-6 on TNF-αproduction in RAW-264.7 Cells.

FIG. 4 is a bar graph representation of the effect of IMS-6 on IL-6production in RAW-264.7 Cells.

FIG. 5 is a bar graph representation of the effect of IMS-6 on humoralresponse in normal and immunocompromised rats.

FIG. 6 is a bar graph representing the effect of IMS-6 on serum cytokinelevels in rats.

FIG. 7 is a bar graph representing the protective effect of IMS-6 in E.coli-induced abdominal species in mice.

FIG. 8 is a bar graph representing the effect of IMS-6 on phagocyticindex in mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the selection and identification of theherbs and obtaining the extract by subjecting the same to solventextraction. The bioassay guided fractionation of the extract to identifythe active markers or active fraction and to develop effective and safecomposition for the use in human beings and animals in immunologicaldisorders as an immunostimulant.

Symplocos racemosa, Roxb, is an evergreen tree or shrub, 6-8.5 m. tall,seen in abundance in the plains and lower hills throughout North andEast India (Chopra, Nayar & Chopra, Glossary Indian Medicinal Plants,Publications and Information Directorate, CSIR, New Delhi, 1956, P.237). The astringent bark of this plant is given for the treatment ofdiarrhoea & dysentery, liver complaints and dropsy. It is also used inophthalmia & conjunctivitis. A decoction of the bark is used to stopbleeding of gums. Bark of this plant is also used for the treatment ofmenorrhagia and other uterine disorders (Sirsi, Indian J. Pharm. 1964,26, 129; Duthie, 11,20; Haines, IV, 521; Dutta & Basu, J. Instn. ChemIndia, 1968, 40, 219; I.P.C. 233; Kirt. & Basu, II, 1511; Dastur,Medicinal Plants, 233.)

Symposide, a new flavan glycoside, which showed antifibrinolyticactivity was isolated from the bark of S. racemosa (Indian J. Chem,1989, 28B, 982.). A new phenolic glycoside named as benzoylsalireposideand known phenol glycoside, salireposide have been isolated from thebark of S. racemosa. These glycosides are reported to show inhibitoryactivity against snake venom Phosphodiesterase type I (Ahmad et al.Phytochemistry, 2003, 63(2), 217-20.

Prosopis glandulosa, Torr, a large shrub or small tree is widelydistributed in the arid parts of the tropical and sub-tropical regionsof the world and widely distributed in India (J. K. Maheshwari, Theflora of Delhi, CSIR, New Delhi, 1963. P. 145). The leaves of the genusProsopis are rich in nutrients, especially nitrogen and useful as greenmanure. Aqueous and alcoholic extract of fresh leaves showed a markedanti bacterial activity against micrococcus pyogenes and basillus coli.(The Wealth of India, Raw materials, Vol. VIII, PID, CSIR, New Delhi,1963, P. 245)

Juliprosopine, an alkaloid exhibiting antibacterial activity wasisolated from the leaves of P. glandulosa (J. Chem. Soc., Pakistan,1982, 4, 285; Chem Abstr. 1983, 98, 122848b). Prosopol, Prosopenol andoleanolic acid were reported from the flowers of P. glandulosa(Fitoterapia, 1986, 57, 457).

EXAMPLE 1 Preparation of Extract from Prosopis glandulosa by PercolationMethod

The shade dried material of leaves of Prosopis glandulosa was pulverizedto coarse powder and about 1 Kg each of powdered material placed indifferent flasks and extracted with n-hexane, dichloromethane,chloroform, ethyl acetate, acetone, ethyl alcohol, methanol, water,chloroform and methanol (1:1), methanol and water (1:1) and ethylalcohol and water (1:1) at room temperature for 24 h to 48 h., thenplant extracts were filtered and concentrated to dryness on rotatoryevaporator or on steam bath at optimum temperature and under reducedpressure.

EXAMPLE 2 Preparation of Extract from Prosopis glandulosa byHot-Soxhalation Method

The coarse powdered material of leaves of Prosopis glandulosa wassubjected to hot-soxhalation using solvents n-hexane, dichloromethane,chloroform, ethyl acetate, acetone, ethyl alcohol, methanol, water,chloroform and methanol (1:1), methanol and water (1:1) and ethylalcohol and water (1:1) at optimum temperature and recycled untilextraction is completed, then plant extracts were filtered andconcentrated to dryness on rotatory evaporator or on steam bath atoptimum temperature.

All extracts such as n-hexane extract (PG-1), dichloromethane extract(PG-2), chloroform extract (PG-3), ethyl acetate extract (PG-4), acetoneextract (PG-5), ethyl alcohol extract (PG-6), methanol extract (PG-7),water extract (PG-8), chloroform:methanol (1:1) extract (PG-9),methanol:water (1:1) extract (PG-10) and ethyl alcohol:water (1:1)extract (PG-11) prepared from the leaves of Prosopis glandulosa by usingpercolation method or hot-soxhalation method were subjected to HPTLC(High Performance Thin Layer Chromatography) and HPLC (High performanceLiquid chromatography) in various mobile phases on precoated TLC plates(Merck) and ODS column for qualitative and quantitative estimation ofmarker compounds and active principles. It was found that the extractsPG-1 to PG-11 were qualitatively and quantitatively similar to eachother.

EXAMPLE 3 Preparation of Extract from Symplocos racemosa by PercolationMethod

The shade dried material of bark of Symplocos racemosa were pulverizedto coarse powder and about 1 Kg of powdered material was placed indifferent flasks and extracted with n-hexane, dichloromethane,chloroform, ethyl acetate, acetone, ethyl alcohol, methanol, water,chloroform and methanol (1:1), methanol and water (1:1) and ethylalcohol and water (1:1) at room temperature for 24 h to 48 h, then plantextract were filtered and concentrated the filtered plant extract todryness on rotatory evaporator or on steam bath at optimum temperatureand under reduced pressure.

EXAMPLE 4 Preparation of Extract from Symplocos racemosa byHot-Soxhalation Method

The coarse powdered material of bark of Symplocos racemosa was subjectedto hot-soxhalation using solvents n-hexane, dichloromethane, chloroform,ethyl acetate, acetone, ethyl alcohol, methanol, water, chloroform andmethanol (1:1), methanol and water (1:1) and ethyl alcohol and water(1:1) at optimum temperature and recycled until extraction is completed,then plant extract were filtered and concentrated the filtered plantextract to dryness on rotatory evaporator or on steam bath at optimumtemperature.

All extracts such as n-hexane extract (SR-1), dichloromethane extract(SR-2), chloroform extract (SR-3), ethyl acetate extract (SR-4), acetoneextract (SR-5), ethyl alcohol extract (SR-6), methanol extract (SR-7),water extract (SR-8), chloroform:methanol (1:1) extract (SR-9),methanol:water (1:1) extract (SR-10) and ethyl alcohol:water (1:1)extract (SR-11) prepared from the bark of Symplocos racemosa by usingpercolation method or hot-soxhalation method were subjected to HPTLC(High Performance Thin Layer Chromatography) and HPLC (High performanceLiquid chromatography) in various mobile phases on precoated TLC plates(Merck) and ODS column for qualitative and quantitative estimation ofmarker compounds and active principles. It was found that the extractsSR-1 to SR-11 were qualitatively and quantitatively similar to eachother.

Screening of Plant Extracts for Immunomodulatory Activity:

The extracts PG-1 to PG-1 and SR-1 to SR-11 were subjected to biologicalscreening in Mouse macrophage (RAW 264.7) cell and fibroblast (L929)cells for in vitro immunomodulatory activity. Following the treatmentwith sub toxic levels of the extract the percent death in the ACDsensitised cells was taken as end point.

The stock solution of the plant extract was prepared in specifiedsolvent at a concentration of 50 mg/ml. The working concentration of 10mg/ml solution was prepared in incomplete Dulbecco's Modified Eagle'sMedium (DMEM) and filter sterilized for further use in the assays.

Mouse macrophage (RAW 264.7) cells and fibroblast (L929) cells were usedin the in vitro assays. These were maintained in DMEM supplemented with10% fetal calf serum (FCS) at 37° C. in a humidified atmospherecontaining 5% CO₂/95% air. The cells were sub cultured upon confluence.

EXAMPLE 5 Assay for Immunostimulatory Effects

Mouse macrophage (RAW 264.7) cells were plated at a cell density of1×10⁵ cells/well in a 96 well micro titer plate. After 24 h ofincubation they were treated with filtered extract (200 μg/ml),Lipopolysaccharide (1 μg/ml) or media alone and incubated for a furtherperiod of 18-24 h. The supernatants as such or diluted ( 1/10^(th) or1/20_(th)) were transferred to pre-incubated (24 h) L929 cells (4×105⁴cells per well). Prior to addition of the supernatant the cells weresensitized with 50 μl of Actinomycin-D (0.33% prepared in DMEM). After24 h of incubation, 20 μl of MTT (5 mg/ml) and 4 hour later 100 μl ofSDS (10%) were added to dissolve the Formazan granules to estimate thecell viability following the transfer of supernatant from the RAW cells.The viability of RAW 264.7 cells (an indicator of extract toxicity) wasestimated by adding MTT after the transfer of its supernatant to L929cells.

EXAMPLE 6 Nitric Oxide (NO) Estimation

Macrophages are part of immune system (innate immunity) whichphagocytose the intruder organism and kill them by release of toxicNitric Oxide (NO). In this experiment, the ability of the plant extractto stimulate the macrophages for NO production was measured as nitritereleased from mouse macrophage cells. Mouse macrophage cells were platedin 96-well culture plates (1×10⁵ cells/well) and incubated for 24 h at37° C. in a humidified atmosphere containing 5% CO₂/95% air. The spentmedia from each well was aspirated and replenished with fresh media andfurther incubated for 48 h with desired concentration of extract inpresence or in absence of Lipopolysaccharide (LPS 1 μg/ml). NOproduction in the supernatant was measured by micro plate assay. Cellsupernatant was mixed with an equal volume of the Griess reagent (1%sulfanilaminde and 0.1% N-[napthyl]ethylenediamine dihydrochloride in2.5% H₃PO₄) at room temperature for 10 min. The absorbance at 540 nm wasdetermined in a microtiter plate reader (Anthos HT II). NO estimationwas carried out using standard curve plotted against known quantity ofsodium nitroprusside. Results presented are in μM concentration obtainedfrom mean OD of triplicate wells of each group.

EXAMPLE 7 Estimation of IL-6 and TNF-∝ Production

This experiment was carried out to know whether the plant extract hasstimulatory effects on the production of proinflammatory cytokines.Mouse macrophage cells, RAW 264.7 were cultured for 48 hours in thepresence or in the absence of filtered plant extract. LPS was used asstimulant for proinflammatory cytokines TNF-∝ and IL-6. Supernatant ofthese samples were harvested immediately for estimations of TNF-∝ andIL-6 by an ELISA as per the manufacturer's protocol and recorded theresults as concentrations in pg/ml.

In Vivo Studies:

Wistar rats/Balb/c mice of either sex were used for the study. Theanimals were housed in standard laboratory conditions and provided witha temperature of 22±3 degree C., relative humidity of 50-55% and a 12 Hrlight/dark cycle. Drinking water and a synthetic pelleted diet (LiptonIndia Ltd, Mumbai) were supplied ad libitum. All experiments wereconducted in strict accordance with NIH guidelines (Guide for the careand use of Laboratory Animals NIH Publication No 86-23, Revised 1985)and under the approval of Institutional ethical committee.

EXAMPLE 8 Effect on Humoral Response and Cytokine Production

Twenty Four Wistar rats of either sex weighing about 200 g each wererandomly grouped into 4 groups of 6 animals each. Animals of group Iremained as normal control and received normal saline orally for 14days. Animals of Group II received IMS-6 (200 mg/Kg body weight) orallyfor 14 days. Animals of group III were similar to control but receivedan immunosuppressive agent. Animals of Group IV were administered IMS-6as above and also received a single dose of Cyclophosphamide(immunosuppressive agent). On the 10^(th) day animals of all groups werechallenged with sheep RBC (SRBC. 5×10⁸ cells/animal) intra peritonially(ip). On the 12^(th) day animals of group III and group IV receivedCyclophosphamide (400 mg/Kg per body weight) intra peritonially (ip). Onday 14, blood was collected from these groups by orbital route andhaemagglunation titer (Antibody levels) in the serum was thenestablished by titration against the SRBC. TNF-∝, IL-2 and IL10 levelswere also estimated to understand the proinflammatory and inhibitorycytokines secreted following the treatment with plant extract.

a. Levels of Immunoglobulin:

For this purpose, serum samples collected from Wistar rats (bothtreatment and control groups) were serially diluted using 0.1% BSAprepared in normal saline in a 96 well “V” bottom plate and were treatedwith an equal volume of 0.1 SRBC (washed at least 3 times to remove theAlsavor's residues). Highest dilution of the serum indicatingagglutination of the SRBC was taken as the endpoint for assessing thelevels of SRBC specific immunoglobulin present in the serum

b. Levels of Cytokines:

The levels of TNF-∝, IL-4 and IL-6 in the serum collected from theanimals treated with extract orally (200 mg/kg) for 14 days weremeasured by an ELISA system as per the manufacture's protocol. Theresults were expressed as concentration in pg/ml both in control and thetreated groups.

EXAMPLE 9 E. coli Induced Abdominal Sepsis

This experiment was conducted in Balb/c mice treated with plant extractat 200 mg/kg body weight orally for 14 days. The treated and untreatedmice were challenged by intra peritoneal administration of 5×10⁸ cellsper ml of pathogenic E. coli. The protection offered by the plantextract in the treated group was measured by comparing with the controlon the incidence of abdominal sepsis and percent mortality.

EXAMPLE 10 Phagocytic Index (PI)

In this experiment the ability of the macrophages to internalise theforeign particle was assessed in presence or in absence of the plantextract. Alveolar macrophages were collected under sterile precautionfrom Balb-c mice (of age 4-5 weeks weighing 20 g each) treated withextract orally (200 mg/kg) for 7 days. Macrophages were washed twicewith DMEM supplemented with 10% FCS and were seeded in a 96-wellmicrotiter plate at 1×10⁶ cells/well and incubated for 24 h at 37° C. ina humidified atmosphere containing 5% CO₂/95% air. The culturedmacrophages were then incubated with the heat killed Candida albicans(10⁸ cells/ml) for a further period of 24 hours. The macrophages werethen stained to assess the Candida albicans particles present in thecytoplasm of macrophages. Number of macrophages containing the ingestedparticles from among the first 200 counted was expressed as thePhagocytic index (PI).

Statistical Analysis:

The data were statistically analysed using One Way Analysis of Varianceusing Bonferroni's Multiple Comparison Test for all parameters exceptfor the E. coli induced abdominal sepsis/mortality where the Chi-Squaretest with Yates correction was adopted. The levels of significance wereexpressed at p<0.05 to p<0.001 levels as per the result of eachanalysis.

The list of extracts screened and their in vitro immunomodulatoryresults are summarised in Tables 1 & 2.

TABLE 1 Proinflammatory Plant extract Cytokine death (%) NO (μM) Control0.00 753.50 ± 3.5  PG-1 26.88 721.00 ± 22.0 PG-2 26.95 745.00 ± 10.5PG-3 26.64 751.00 ± 38.0 PG-4 36.29 748.50 ± 34.5 PG-5 36.85 743.50 ±0.5  PG-6 18.90 716.50 ± 26.5 PG-7 5.09 843.00 ± 2.0  PG-8 61.25 1223.00± 29.0* PG-9 18.25 752.00 ± 1.5  PG-10 53.30  871.50 ± 26.50 PG-11 33.30760.50 ± 2.5  *P < 0.001 as compared to control. Values are expressed asmean ± SEM (n = 2).

TABLE 2 Proinflammatory Plant extract Cytokine death (%) NO (μM) Control0.00 753.50 ± 3.5 SR-1 19.10 707.00 ± 15.5 SR-2 15.20 736.00 ± 5.00 SR-320.10 749.00 ± 9.0 SR-4 58.50 698.00 ± 44.5 SR-5 40.10 751.00 ± 3.0 SR-620.20 546.50 ± 21.5 SR-7 16.10 748.00 ± 108.0 SR-8 67.30 788.00 ± 11.0SR-9 57.90 745.00 ± 3.5 SR-10 66.70 652.00 ± 1.5 SR-11 14.30 648.00 ±28.0 P values are not significant as compared to control. Values areexpressed as mean ± SEM (n = 2)

The above in vitro results demonstrate the immunomodulatory activity ofextracts PG-8 and SR-8 more potent than the other extracts and alsodevoid of any toxicity. These two short listed extracts viz. PG-8 andSR-8 were selected for further study of immunostimulatory activity. Asthe objective of the present invention is to formulate a naturalimmunostimulant composition, which is effective and free of undesirableside effects, the various combinations of these extracts were preparedand subjected to in vitro immunostimulatory activities for potentcombination of herbal composition for immunostimulatory activity. Thevarious combinations of the extracts PG-8 and SR-8 were prepared in theratio of PG-8: SR-8 (1:9) extract (IMS-1), PG-8:SR-8 (9:1) extract(IMS-2), PG-8: SR-8 (1:3) extract (IMS-3), PG-8: SR-8 (3:1) extract(IMS-4), PG-8: SR-8 (1:4) extract (IMS-5), PG-8: SR-8 (1:1) extract(IMS-6) and PG-8: SR-8 (4:1) extract (IMS-7).

The in vitro immunomodulatory activity results of various combinationsof PG-8 and SR-8 are shown in Table 3

TABLE 3 Proinflammatory Extract Cytokine death (%) NO (μM) Control 0.00753.50 ± 3.5  IMS-1 78.17 846.10 ± 15.3  IMS-2 78.17 798.07 ± 48.0 IMS-3 82.00 1129.27 ± 21.0*  IMS-4 76.00 943.00 ± 71.80 IMS-5 84.60946.15 ± 53.80 IMS-6 88.00 1301.28 ± 91.0** IMS-7 85.00 784.60 ± 46.15**P < 0.01 as compared to control. *P < 0.05 as compared to control.Values are expressed as mean ± SEM (n = 2)

The above table demonstrates the combination extract IMS-6 has highstimulation ability of proinflammatory cytokines from the macrophages,ability to stimulate the production of significant levels of NO andmoderate to high level stimulation of the innate and acquired immunityand absolutely safe without any undesirable side effects.

Immunostimulatory Effects of IMS-6:

In the present study, Immunostimulatory effects of plant extract IMS-6,to produce proinflammatory cytokines TNF-∝ and IL-6 were assessed. Invitro study was conducted in mouse macrophage cells (RAW 264.7) and infibroblast cells (L929). The levels of macrophage activation (phagocyticactivity), cell mediated and humoral responses were some of the otherparameters investigated to assess the immunostimulatory effects of theproduct. In vivo and ex-vivo studies were also conducted to assess thelevels of cytokines produced in response to the plant extract treatment.

Assay for Immunostimulatory Effects:

The assay conducted in the cell lines indicated that the supernatant ofthe macrophage cells were able to successfully cause more than 50% deathin the ACD sensitized L929 cells (end point) comparable to the levels inLPS group. The control group however did not exhibit the same levels ofdamage. Further, MTT reduction assay indicated that the plant extract byitself was not toxic to the cells. The significantly higher levels ofthe death in L929 cells (p<0.001) in the IMS-6 treated group compared tocontrol following the transfer of supernatant from RAW cells was not onaccount of IMS-6 toxicity but due the cytokines released from theactivated macrophages. Correspondingly the treatment group showedsignificantly lower levels (p<0.001) of survival compared to control.The higher levels of death in L929 cells indicate that IMS-6 was able tostimulate the macrophage to produce the proinflammatory cytokines. Theseresults are shown in Table 4 and FIG. 1.

TABLE 4 Effect of IMS-6 on Proinflammatory cytokine induced cell deathin ACD sensitized L929 cells: Proinflammatory Cytokine Treatment Groupinduced Cell Death (%) Control 0 LPS 79.0 IMS-6 87.11 IMS-6 + LPS 87.66

Nitric Oxide (NO) Estimation:

This experiment indicated that the macrophages were activated by theplant extract to produce nitric oxide to the levels comparable to LPSgroup, indicating that the plant extract was able to stimulate theinnate immune system of the body and thus help preventing the invadingorganisms. Results are shown in Table 5 and FIG. 2.

TABLE 5 Groups NO Levels (μM) Control 800 ± 50 LPS 2862.50 ± 87.50*IMS-6 2150.00 ± 50.00* IMS-6 + LPS 2850.00 ± 50.00* *p < 0.001 ascompared to control

The nitrite release in the IMS-6 treated group (2150±50 μM) wassignificantly higher (p<0.001) compared to control (800±50 μM). Thegroup which received LPS along with the plant extract IMS-6 recorded NOlevels of 2850±150 μM and was also significantly higher (p<0.001)compared to control.

Estimation of TNF-∝ and IL-6 Production:

In vitro cytokine estimation in the supernatant of macrophage cellsindicated high levels of proinflammatory cytokines in the IMS 6 treatedgroup compared to the control. The levels of TNF-∝ were significantlyhigher (p<0.01) in the IMS 6 treated groups compared to the control.Further, the TNF-∝ levels were significantly higher (p<0.001) in the IMS6 and LPS combination group indicating the synergistic effect on accountof IMS 6. Treatment of RAW cells with the IMS 6 for 24 produced anincrease in the levels of unbound TNF-α (2703.00±3.70 pg/ml) compared tocontrol (1662.00±3.50 pg/ml). The levels in groups treated with IMS-6and LPS also showed a substantial increase (3516.00±24.50 pg/ml)compared group treated with LPS alone (3432.00±140.50 pg/ml). Resultsare shown in Table-6 and FIG. 3.

TABLE 6 Groups TNF ∝ Levels (pg/ml) Control 1662.00 ± 3.50 LPS 3432.00 ±140.50^(*) IMS 6 2703.00 ± 3.70* IMS6 + LPS 3516.00 ± 24.50^(*)

The levels of IL-6 in the IMS-6 groups were higher than the control andexhibited the synergistic effect in the group treated with IMS-6 alongwith LPS and was significantly higher (p<0.001) compared to the groupstreated with LPS alone. Results are shown in Table 7 and FIG. 4.

TABLE 7 Groups IL-6 Conc. (pg/ml) Control  542 ± 4 LPS* 11187 ± 214IMS-6  760 ± 6.8 IMS-6 + LPS 21254 ± 10.4

Effect on Humoral Response and Cytokine Production:

Wistar rats treated with the IMS-6 showed significantly higherhemagglutination titer (HA) (p<0.01) compared to control where the meanvalues stood at 3.278±0.181 only. The group of animals challenged withSRBC and subsequently with the cyclophosphamide did not elicit anyimmune response and had significantly lower (p<0.01) antibody titercompared to control animals. The immune suppressant activity of thecyclophosphamide was completely overcome in the animals treatedsimultaneously with IMS6 where the antibody response stood atsignificantly higher levels (p<0.001) compared to the Cyclophosphamidegroup indicating that IMS-6 was able to restore the HA titer in immunesuppressed animals. Results are shown in Table 8 and FIG. 5.

TABLE 8 Groups Antibody titre Control 3.278 ± 0.181 IMS-6 7.833 ± 0.114*Cyclophosphamide 0.444 ± 0.141* Cyclophosphamide + IMS-6 3.333 ±0.31^(*) Error bar represents the mean ± SEM of mean values of threeexperiments where *P < 0.01 as compared to control and *p < 0.001 ascompared to cyclophosphamide.

Levels of Cytokines:

The studies conducted in Wistar rats indicated that the IMS-6 treatedanimals had significantly higher levels (p<0.001) of proinflammatorycytokines TNF-∝, IL-6 and IL-4 compared to the control animals. Further,the levels immunosuppressive cytokine IL-10 was at not detectable range(data not provided) compared to the control group. This result suggeststhat the plant extract was able to stimulate the immune system torelease the proinflammatory responses. Results are shown in Table 9 andFIG. 6.

TABLE 9 Groups Control* IMS-6* TNF-α 87.07 ± 2.72 112.92 ± 1.36 IL-6170.65 ± 22.65 225.55 ± 3.33 IL-4 48.66 ± 1.3   58.33 ± 1.66 Error barrepresents the mean ± SEM of mean values from three differentexperiments where n = 10. * Values are in pg/ml *p < 0.001 as comparedto control.E. coli Induced Abdominal Sepsis:

The study conducted on the incidence of abdominal sepsis and mortalityin the E. coli challenged mice treated with IMS-6 showed significantlyhigher levels of protection (p<0.001) compared to control. While theextent of mortality in the IMS-6 treated group was on 20%, the same inthe control was 100% following a 24-hour challenge period. Further theanimals treated with IMS-6 were able to survive beyond 5 days suggestingthe activation of cell-mediated immunity in this group. The bloodsamples collected from the animals when cultured on an agar plate showedlower colony count in the IMS-6 treated animals compared to the controlanimals. Results are shown in FIG. 7.

Phagocytic Index:

Alveolar macrophages collected from IMS-6 treated mice indicated a highdegree of protective activation of innate immune system. This wasevident due to the enhanced ability of the macrophages to phagocytosethe interloper in an ex-vitro experiment detailed earlier. A total of120 macrophages from out of 200 macrophages counted indicated thepresence of the engulfed Candida albicans particles in the IMS-6 treatedgroup which was significantly higher (p<0.001) compared to the controlwherein only 44 of them showed the active phagocytosis. Results areshown in FIG. 8.

The alveolar macrophages collected from the mice treated with the IMS-6thus exhibited high efficiency in internalising the Candida albicansparticles.

EXAMPLE 11 Bioassay Guided Fractionation of Active Extract IMS-6

About 100 g of the combined extract (IMS-6) is macerated with methanolto give methanol soluble fraction (IMS-6A) and methanol insolublefraction. The methanol insoluble extract was further fractionated intomethanol:water (10%) IMS-6B, methanol:water (25%) IMS-6C, methanol:water(50%) IMS-6D, methanol-water (75%) IMS-6E and water (100%) IMS-6Ffractions. All fractions IMS-6A to IMS-6F were shown immunostimulatingactivity and the results are given in Table 10. The methanol solublefraction IMS-6A and methanol:water (10%) fraction, IMS-6B have found tobe more potent. The methanol soluble fraction was taken up for furtherdetailed study of identification of marker compounds and activeprinciples responsible for immunostimulating activity.

EXAMPLE 12 Column Chromatography of IMS-6A

About 25 g of IMS-6A fraction was subjected to column chromatographyover silica gel (60-120 mesh) and eluted with increasing gradient ofhexane, hexane and ethyl acetate solvent mixture, ethyl acetate andethyl acetate and methanol solvent mixture. Twenty two fractions of 250ml each were collected and mixed after analysing TLC over precoated TLCplates. The semi purified fractions were tested for the presence ofAlkaloids, Glycosides, Amino Acids, Amino glycosides, polysaccharides,flavonoids and Tannins. The semi purified fractions IMS-6A1 to IMS-6A12were subjected to in vitro immunomodulatory activity, and results aresummarized in Table 11.

TABLE 10 Proinflammatory cytokine Fraction death (%) NO (μM) Control0.00 458.00 ± 3.5 IMS-6A 88.0 874.00 ± 25.0* IMS-6B 78.0 809.00 ± 4.5*IMS-6C 52.2 695.50 ± 45.5** IMS-6D 47.2 732.00 ± 18.0* IMS-6E 46.5722.00 ± 16.5* IMS-6F 55.1 683.50 ± 24.5** *P < 0.001 as compared tocontrol. **P < 0.01 as compared to control. Values are expressed as mean± SEM (n = 2).

TABLE 11 Proinflammatory cytokine Fraction death (%) NO (μM) Control0.00 750.50 ± 0.5 IMS-6A1 57.6 752.00 ± 2.0 IMS-6A2 53.4 711.00 ± 1.0IMS-6A3 52.8 750.50 ± 0.5 IMS-6A4 56.4  732.00 ± 20.0 IMS-6A5 57.9749.50 ± 4.5 IMS-6A6 75.8  907.50 ± 7.5* IMS-6A7 55.8 759.50 ± 0.5IMS-6A8 56.2 707.50 ± 7.5 IMS-6A9 76.2  917.50 ± 17.5* IMS-6A10 87.8 1325.00 ± 25.0* IMS-6A11 50.3 763.00 ± 5.0 IMS-6A12 57.1 752.00 ± 2.0*P < 0.001 as compared to control.

The bioassay guided fractionation and purification of IMS-6 extractdemonstrate the bioactivity in IMS-6A and its semi purified fractionIMS-6A10 in addition to all other fractions and its semi purifiedfractions that have shown moderate to good activity. It is clear fromthe above two tables that the activity of IMS-6 could be due to thepresence of many active compounds which shows synergic action as a wholein IMS-6 extract. It is therefore the present invention is unique andnovel for combining in a particular ratio of two herbal extracts whichis superior than individual extracts and its fractions.

Process for preparation of Pharmaceutical Formulations comprisingextract of plants Symplocos racemosa and Prosopis glandulosa andpharmaceutically acceptable carriers to provide different deliverysystems.

EXAMPLE 13 Preparation of IMS-6 Syrup

Sl. Formula Formula Formula No. Name of Ingredient, Formula I II III IVFormula V 1 IMS - 6 extract IH 50 mg 100 mg 125 mg 250 mg 500 mg 2 SUGARD 30 (sucrose)/ 3.4 gm 3.4 gm 3.4 gm 3.5 gm 3.75 gm IP 3 Citric acid IP0.01 mg 0.01 mg 0.01 mg 0.02 mg 0.02 mg 4 Methyl paraben sodium 0.01 mg0.01 mg 0.01 mg 0.01 mg 0.01 mg IP 5 Propyl paraben sodium 0.0025 mg0.0025 mg 0.0025 mg 0.0025 mg 0.0025 mg IP 6 Strawberry flavor IFF 0.005mg 0.005 mg 0.005 mg 0.005 mg 0.005 mg 7 D M water Qs to 5 ml Qs to 5 mlQs to 5 ml Qs to 5 ml Qs to 5 ml (demineralised water) IP

Process for Preparation:

First sugar was dissolved with DM Water in a jacketed vessel, thenextract was added into the solution and mixed for 10-15 min. and theresultant was filtered through Polypropylene pad into another jacketedvessel, then citric acid was dissolved in small quantity of DM(demineralised) water and mixed with the resultant, methyl parabensodium and propyl paraben sodium was dissolved in small quantity of DM(demineralised) water and mixed with the resultant mixture at 60° C.-70°C. and then the mixture was cooled, flavor was added at 40° C. or lessand mixed for 5-10 min. Then the volume was maintained, and mixed for10-15 min. and filtered to a clean storage vessel through Polypropylenepad (10 micron).

EXAMPLE 14 Preparation of IMS-6 Tablets

Sl Formula Formula Formula Formula No. Name of Ingredient, Formula I IIIII IV Formula V VI 1 IMS-6 extract IH  50 mg 100 mg 150 mg 200 mg 250mg 500 mg 2 Microcrystalline 450 mg 350 mg — — 100 mg 100 mg celluloseIP 3 P. G. STARCH —  50 mg  50 mg — — — (pregelatinised starch) IP 4Lactose IP — — 300 mg  50 mg 100 mg 150 mg 5 Dibasic calcium — — — 250mg  50 mg 200 mg phosphate IP 6 D M water Q.S. Q.S. Q.S. Q.S. Q.S. Q.S.(demineralised water) IH

Process for Preparation:

Formula I: Microcrystalline cellulose IP (Passed through Sieve No. 60)was loaded in a suitable mixer and granulated with IMS-6 extract IH andDM (demineralised) Water. The wet mass was passed through Sieve No. 8and dried in suitable drier till the moisture content of 2-4%. The driedmass was passed through Sieve No. 16 and the lot was mixed uniformly.Formula II: Microcrystalline cellulose IP and P. G. STARCH IP (Bothpassed through Sieve No. 60) were loaded in a suitable mixer and mixedfor 5 mins. and granulated with IMS-6 extract IH and DM (demineralised)Water. The wet mass was passed through Sieve No. 8 and dried in suitabledrier till the moisture content of 2-4%. The dried mass was passedthrough Sieve No. 16 and the lot was mixed uniformly.Formula III: P. G. STARCH IP (pregelatinised starch) and Lactose IP(Both passed through Sieve No. 60) were loaded in a suitable mixer andmixed for 5 mins. and granulated with IMS-6 extract IH and DM(demineralised) Water. The wet mass was passed through Sieve No. 8 anddried in suitable drier till the moisture content of 2-4%. The driedmass was passed through Sieve No. 16 and the lot was mixed uniformly.Formula IV: Lactose IP and Dibasic calcium phosphate IP (Both passedthrough Sieve No. 60) were loaded in a suitable mixer and mixed for 5mins. and granulated with IMS-6 extract IH and DM (demineralised) Water.The wet mass was passed through Sieve No. 8 and dried in suitable driertill the moisture content of 2-4%. The dried mass was passed throughSieve No. 16 and the lot was mixed uniformly.Formula V: Microcrystalline cellulose IP, Lactose IP and Dibasic calciumphosphate IP (Both passed through Sieve No. 60) were loaded in asuitable mixer and mixed for 5 mins. and granulated with IMS-6 extractIH and DM (demineralised) Water. The wet mass was passed through SieveNo. 8 and dried in suitable drier till the moisture of 2-4%. The driedmass was passed through Sieve No. 16 and the lot was mixed uniformly.Formula VI: Microcrystalline cellulose IP, Lactose IP and Dibasiccalcium phosphate IP (Both passed through Sieve No. 60) were loaded in asuitable mixer and mixed for 5 mins. and granulated with IMS-6 extractIH and DM (demineralised) Water. The wet mass was passed through SieveNo. 8 and dried in suitable drier till the moisture of 2-4%. The driedmass was passed through Sieve No. 16 and the lot was mixed uniformly.

Pharmaceutical Ingredients for Tablet Compression (Formula I to VI)

Formula I to V Formula VI Sl. No. Name of Ingredient Mg/Tablet Mg/Tablet1 IMS-6 granules IH 500.00 950.00 2 Sodium starch Glycolate IP 30.0030.00 3 Calcium carbonate IP 14.00 14.00 4 CABOSIL M5 (colloidal 3.003.00 silicon dioxide) IP/USP 5 Magnesium stearate IP 3.00 3.00 Total550.00 1000.00

Procedure of Compression:

Sodium starch Glycolate IP, Calcium carbonate IP and CABOSIL M5(colloidal silicon dioxide)IP/USP were mixed and passed through SieveNo. 60 and blended in a suitable mixer with IMS-6 granules IH for 5min., Magnesium stearate IP was passed through Sieve No. 60 and blendedwith the above for 3 min. The blend was ready for tablet compression.

Tooling: Caplet Shape, Round Shape, Oval Shape, and Triangular Shapeetc. EXAMPLE 15 Preparation of IMS-6 Capsules

Sl Formula Formula Formula No. Name of Ingredient, Formula I II III IVFormula V 1 IMS - 6 extract IH  50 mg 100 mg 150 mg 200 mg 250 mg 2Micro crystalline cellulose IP 250 mg 150 mg — — 100 mg 3 P. G. STARCH(pregelatinised —  50 mg  50 mg — — starch) IP 4 Lactose IP — — 300 mg 50 mg 100 mg 5 Dibasic calcium phosphate IP — — — 250 mg  50 mg 6 D M(demineralised) water IH Q.S. Q.S. Q.S. Q.S. Q.S.

Process for Preparation:

Formula I: Microcrystalline cellulose IP (Passed through Sieve No. 60)was loaded in a suitable mixer and granulated with IMS-6 extract IH andDM (demineralised)Water. The wet mass was passed through Sieve No. 8 anddried in suitable drier till the moisture content of 2-4%. The driedmass was passed through Sieve No. 16 and the lot was mixed uniformly.Formula II: Microcrystalline cellulose IP and P. G. STARCH IP (Bothpassed through Sieve No. 60) were loaded in a suitable mixer and mixedfor 5 min. and granulated with IMS-6 extract IH and DM (demineralised)Water. The wet mass was passed through Sieve No. 8 and dried in suitabledrier till the moisture content of 2-4%. The dried mass was passedthrough Sieve No. 16 and mixed uniformly.Formula III: P. G. STARCH IP and Lactose IP (Both passed through SieveNo. 60) were loaded in a suitable mixer and mixed for 5 min. andgranulated with IMS-6 extract IH and DM (demineralised) Water. The wetmass was passed through Sieve No. 8 and dried in suitable drier till themoisture content of 2-4%. The dried mass was passed through Sieve No. 16and mixed uniformly.Formula IV: Lactose IP and Dibasic calcium phosphate IP (Both passedthrough Sieve No. 60) were loaded in a suitable mixer and mixed for 5min. and granulated with IMS-6 extract IH and DM (demineralised) Water.The wet mass passed through Sieve No. 8 and dried in suitable drier tillthe moisture content of 2-4%. The dried mass was passed through SieveNo. 16 and mixed uniformly.Formula V: Microcrystalline cellulose IP, Lactose IP and Dibasic calciumphosphate IP (all passed through Sieve No. 60) were loaded in a suitablemixer and mixed for 5 min. and granulated with IMS-6 extract IH and DM(demineralised) Water. The wet mass was passed through Sieve No. 8 anddried in suitable drier till the moisture content of 2-4%. The driedmass was passed through Sieve No. 16 and mixed uniformly.

Capsule Filling Formula:

Formula I to II Formula III to V Sl. No. Name of Ingredient, Mg/CapsuleMg/Capsule 1 IMS-6 granules IH 300.00 500.00 2 CABOSIL M5 (colloidal2.00 2.00 silicon dioxide) IP/USP 3 Magnesium stearate IP 3.00 3.00Total 305.00 505.00Description of capsule: Size ‘0’ and ‘00’ Clear transparent/or coloredempty hard gelatine and/or Vegetable (HPMC) capsules.

Immune stimulators are of great value in the treatment of Cancerconditions, Hepatitis B, HIV and in other immunity breakdown situationscommonly associated with the bacterial and viral diseases.

Anticancer drugs generally inhibit the cell proliferation and bringabout the apoptosis of the cancerous cells. Combination of anticancerdrugs with the Immunostimulatory drugs such as IMS-6 in the presentinvention is of added advantage due to the surge in proinflammatorycytokine TNF-α which hastens the process of apoptosis of the flaggedcancerous cells. Further, enhanced non-specific immune response underthe influence of the immune potentiators will lead to the increasedmacrophage activity thereby hastening the process of phagocytosis of theapoptosed cells and help in clearing the debris. The immunostimulant,IMS-6 may be given as adjuvant dose ranging from 250 mg to 500 mg in theform of syrup, tablet, capsule etc. in cancer patients along withprescribed anticancer drugs for the above immunostimulatory benefits.

In HIV, the cell-mediated immunity is affected due to consistent deathof cells CD4+ and CD8+ cells and is characterized by reduced count ofthese cells responsible for fighting against the invading pathogen.IMS-6 with its Immunostimulatory properties will be of help in suchsituations to increase the cell-mediated immunity by initiating theclonal expansion of the lymphocytes. IMS-6 may be used in AIDS patientsas syrup, tablet, and capsule etc. consisting of active dose of 250 mgto 1.0 g.

In situations of Hepatitis B and other viral infections, a potentantiviral drug with an Immunostimulatory agent like IMS-6 in the form ofsyrup, tablet, capsule etc. dose ranging from 250 mg to 500 mg will helpin increasing the cell-mediated response and Humoral response to combatthe viral infections and help in scavenging of the affected cells.Further, the enhanced levels of non-specific and specific immunity willalso help in preventing the possibility of co-infection with bacterialor other viral agents.

While this invention has been described in detail with reference tocertain preferred embodiments, it should be appreciated that the presentinvention is not limited to those precise embodiments. Rather, in viewof the present disclosure, which describes the current best mode forpracticing the invention, many modifications and variations wouldpresent themselves to those skilled in the art without departing fromthe scope and spirit of this invention.

1. A method for treating immunodeficiencies in animals including humanbeings by administering to a patient, natural immunostimulantcomposition comprising a therapeutically effective amount of extracts ofplants Symplocos racemosa and Prosopis glandulosa in a pharmaceuticallyacceptable carrier or otherwise.
 2. The method according to claim 1,further comprising invoking a cell mediated immune response in patientsby administering the natural immunostimulant composition comprising atherapeutically effective amount of extracts of plants Symplocosracemosa and Prosopis glandulosa in a pharmaceutically acceptablecarrier or otherwise.
 3. The method according to claim 1, furthercomprising invoking humoral antibody response in patients byadministering the natural immunostimulant composition comprising atherapeutically effective amount of extracts of plants Symplocosracemosa and Prosopis glandulosa in a pharmaceutically acceptablecarrier or otherwise.
 4. The method according to claim 1, furthercomprising treating immunological disorders including AIDS, Hepatitis,Cancer, and any relapse conditions thereof by administering the naturalimmunostimulant composition comprising a therapeutically effectiveamount of extracts of plants Symplocos racemosa and Prosopis glandulosain a pharmaceutically acceptable carrier or otherwise.
 5. The methodaccording to claim 1, further comprising administering to a patient anatural immunostimulant composition comprising a therapeuticallyeffective amount of extracts of plants Symplocos racemosa and Prosopisglandulosa in a pharmaceutically acceptable carrier or otherwise forstimulating both specific and non-specific immune reaction.
 6. Themethod according to claim 1, further comprising administering to apatient a natural immunostimulant composition comprising atherapeutically effective amount of extracts of plants Symplocosracemosa and Prosopis glandulosa in a pharmaceutically acceptablecarrier or otherwise for enhancing responsiveness of macrophages andsubsets of T-lymphocytes and B-lymphocytes involved in antibodysynthesis.
 7. The method according to claim 1, further comprisingadministering to a patient a natural immunostimulant compositioncomprising a therapeutically effective amount of extracts of plantsSymplocos racemosa and Prosopis glandulosa in a pharmaceuticallyacceptable carrier or otherwise for enhancing 4 fold (1:256) increase inthe primary antibody titre.
 8. The method according to claim 1, furthercomprising administering to a patient a natural immunostimulantcomposition comprising a therapeutically effective amount of extracts ofplants Symplocos racemosa and Prosopis glandulosa in a pharmaceuticallyacceptable carrier or otherwise to cause an increase by 60% inphagocytosis.
 9. The method according to claim 1, further comprisingadministering to a patient a natural immunostimulant compositioncomprising a therapeutically effective amount of extracts of plantsSymplocos racemosa and Prosopis glandulosa in a pharmaceuticallyacceptable carrier or otherwise for stimulating proinflammatorycytokines IL-6 at the rate of 1.4 times and TNF-α at the rate of 1.6times in mouse macrophage and fibroblast cell lines and for suppressinginhibitory cytokines IL-10 in mice.
 10. A method of producing a naturalimmunostimulant composition, the method comprising extracting Prosopisglandulosa leaves by percolation, filtering the plant extract,concentrating the plant extract to dryness on rotatory evaporator or onsteam bath at optimum temperature and producing a herbal compositioncomprising the said dry extract and a pharmaceutically acceptablecarrier.
 11. A method of producing a natural immunostimulantcomposition, the method comprising extracting plant extract fromProsopis glandulosa by hot soxhalation, filtering the plant extract,concentrating the plant extract to dryness on rotatory evaporator or onsteam bath at optimum temperature and producing a herbal compositionemploying the said dry extract and a pharmaceutically acceptablecarrier.
 12. A method of producing a natural immunostimulantcomposition, the method comprising extracting plant extract fromSymplocos racemosa by percolation, filtering the plant extract,concentrating the plant extract to dryness on rotatory evaporator or onsteam bath at optimum temperature and producing a herbal compositionemploying the said dry extract and a pharmaceutically acceptablecarrier.
 13. A method of producing a natural immunostimulantcomposition, the method comprising extracting plant extract fromSymplocos racemosa by hot soxhalation, filtering the plant extract,concentrating the plant extract to dryness on rotatory evaporator or onsteam bath at optimum temperature and producing a herbal compositionemploying the said dry extract and a pharmaceutically acceptablecarrier.
 14. A method for obtaining the active fraction of extracts ofplant Symplocos racemosa and plant Prosopis glandulosa by subjectingsaid extract to bioassay-guided fractionation employing methanol solublefraction, methanol and water (90:10) soluble fraction, methanol andwater (75:25) soluble fraction, methanol and water (50:50) solublefraction, methanol and water (25:75) soluble fraction and water-solublefraction.
 15. A method for obtaining the active fraction of extracts ofplant Symplocos racemosa and plant Prosopis glandulosa by subjecting theextract to bioassay-guided fractionation employing methanol solublefraction and methanol:water (90:10) fraction.